INTRODUCTION:

The medicinal plants have been used for ages as remedies for human diseases. Today there is an incessant and imperative necessity to find out new antibacterial compounds with various chemical structures and new mechanisms of action because there has been a shocking increase in the prevalence of new and reemerging contagious syndromes. This is why ethnobotany and ethnopharmacology are working to identify, all over the world, plants that are known to be active and for which it is up to modern research to specify the properties and validate their use¹. The search for new ones molecules must be undertaken within plant biodiversity using ethnopharmacological data. An increasing number of reports dealing with the evaluation of the antimicrobial effects of different extracts of various medicinal plants are frequently available.^2-8However, due to the emergence of new strains of bacteria and weak chemotherapeutic agents and antibiotic resistance of pathogens, several medicinal plants have been tested for their potential antimicrobial activity^9-14. This has led us to focus more on the study of medicinal plants such as Pergularia tomentosa L.

Pergularia tomentosa L., Commonly known as Ghalka in Algeria, is perennial shrub about 50-60 cm high, reaching 1m in good conditions^15-17. It is poisonous plant that is known to be distributed in the Saharian and Sub-Saharian countries of North Africa¹⁸including Algeria, Niger, and Egypt¹⁹. This plant is also common in the Middle-East region including Saudi Arabia²⁰ and Jordan²¹. The plant is known to be as producing corrosive white latex that may severely harm the skin.

In spite of being poisonous, Pergularia tomentosa is used extensively in traditional medicine by North Saharian and SubSaharian populations. A decoction of the leaves and stems is used for the treatment of bronchitis and tuberculosis, a medication that should be taken with great care and is forbidden for pregnant women²². The plant was reported to have molluscidal activity²³ and persistent hypoglyceaemic effects¹⁹. The roots are used for the treatment of bronchitis, constipation and skin diseases²⁴. Several reports mentioned this plant which used for the treatment asthma and as antirheumatic agent^24-25.

The isolation and characterization of two new triterpenes along with five other known compounds was reported already for the first time from Pergularia tomentosa²⁶ Now we wish to report the study and evaluation of the antibacterial and antioxidant activities of Pergularia tomentosa L. against several Gram-positive and Gram-negative bacterial strains in vitro.

MATERIAL AND METHODS:

Chemicals and reagents:

The aerial parts of Pergularia tomentosa L were collected randomly from the Ouargla desert, south of Algeria in April 2016. The medicinal plant was deposited at Dynamics, Interaction and Systems Reactivity Laboratory, Process Engineering Department, Faculty of Applied Sciences, University Kasdi Merbah, Ouargla. Fresh plant material was washed under running tap water, air dried under dark and then homogenized to fine powder using an electrical mixer "Panasonic Type" for 20 minutes, and stored in closed container away from light and moisture.

Preliminary Phytochemical Analysis:

The preliminary phytochemical analysis of the crude powder of Pergularia tomentosa L showed that this plant contains mainly many active ingredients: volatile oils, terpenes and alkaloids, one of the antioxidants of the bacteria responsible for the effect of microbs, also contains flavonoids including glycosides antioxidant, phenols and saponins. As for the nature of the extracts were characterized by strength viscous dark green color and aromatic smell, due to the emergence of green chlorophyll pigment and material xanthine.

Extraction of plant material:

The extracts were prepared by soaking 200g of the plant powder in a mixture of EtOH/H₂O (70/30) for 24 hours. The procedure was repeated three times and the filtrates were combined before being evaporated under reduced pressure. The resulting extracts were diluted with distilled water and left overnight. The filtrates were subjected to extraction by various solvents with increasing polarity (methylene chloride, ethyl acetate and butanol). The organic phases were separated and evaporated and the resulting residues were stored at 4°C.

Microorganisms

All bacterial standard strains: Escherichia coli G(-), pseudomonas aeruginosa G(-), and (Streptococcus aureus) G(+) were obtained from de Biotechnologie Laboratory « INRAP » (Institut National de la Recherché et d’Analyse Physico-chimique) of Tunisie.

Preparation of the bacterial culture media

3.7 of muller Hilton agar were mixed with hot distilled water and autoclaved at 121°C and 2 atm for 15 minutes. After autoclaving, it was allowed to cool to 45°C in a water bath. Then the medium was poured into sterilized Petri dishes with a uniform depth of approximately 5 mm²⁷.

Preparation of plant extract impregnated discs

Whatman N°1 filter paper was used to prepare discs of 6 mm in diameter. They were sterilized by autoclaving and then dried during the autoclaving cycle. The discs were then impregnated with extract of the plants²⁸.

Disc diffusion method

Disc diffusion method for antimicrobial susceptibility test was carried out according to the standard method by Kirby-Bauer to assess the presence of antibacterial activities of plant extracts^28-29. The resulting residue of all extracts stored at 4°C was tested at concentrations 10^-1 g/ml and were prepared in DMSO.

Standard antimicrobs:

A standard antimicrobs: Ampicillin/Nystatine were obtained from Italian company “Liofilchem.

Antioxidant activity evaluation:

The oxidative stress resulting from the imbalance created by the excessive production of reactive oxygen species (ROS) is considered to be critically involved in the normal aging process but also in the development and progression of various human pathologies, including cancers. Indeed, initiation and progression of cancer have been associated with oxidative stress by increasing DNA mutations or inducing DNA damage, genome instability, and cell proliferation. Normal cells are hypersensitive to reactive oxygen species (ROS) if they are not sufficiently protected by antioxidant mechanisms and can lead to cancer formation²⁹ (Harbone et al., 1984).

The evaluation of antioxidant activity in vitro is done by several techniques. These methods are based exclusively on the reducing capacity or trapping of radicals as an indicator of its antioxidant potential.

The percentage inhibition (% I) formula:

The estimate of the free radical activity is expressed as the percentage inhibition value (% I) calculated using the following formula:

% I = [(Abs₀ – Abs₁)/Abs₀)] x100

With

Abs₀: absorbance of analytical white.

Abs₁: absorbance of the solution in the presence of extract.

The curve giving the variation of (%I) as a function of the different concentrations of the extract makes it possible to determine the antiradical activity or IC50 (Efficient Concentration 50%), defined as the quantity of extract necessary to halve the initial concentration of DPPH.

Antioxidant activity by using DPPH test:

The demonstration of the in vitro antioxidant activity of the compounds tested was carried out by trapping the free radical DPPH. The antioxidant capacity is expressed in Tocopherol (VE) or Ascorbic acid (VC) equivalents.

Tocopherol (VE) and Ascorbic acid (VC) are antioxidants, which are used in biology and biochemistry to limit damage due to oxidative stress.

The antiradical activity of DPPH^∙ was determined based on the assays described by Brand-Williams with some modifications. Thus, in a volume of 1ml, different concentrations of the test extract are prepared in methanol, then 2ml of the solution of DPPH. 0.1mM concentration. After vigorous stirring, the mixture is incubated for 1 hour in the dark and at room temperature, and then the absorbance is measured at 515 nm by a UV vis spectrophotometer (Jasco-V530). A solution containing 1ml methanol and 2ml of DPPH. Considered as analytical white is prepared in parallel.

FRAP test

(Ferric Reducing/Antioxydant power) is an antioxidant test that studies the effectiveness of reactive antioxidants in chromatography reaction that is studying the ability of extracts as inhibitors of oxidation process. The method principle depends on the coloring or non-coloring of the complex (TPTZ) in the acid medium^31-32.

RESULTS AND DISCUSSION

Anti bacterial activity

Three clinical antibiotic-isolates, Escherichia coli ATCC 8739 G(-),Pseudomonas aeruginosa G(-) and Staphylococus aureus ATCC 6538 G(+) were selected for the antibacterial analysis. As shown, table 1 summarized the microbial growth inhibition and clarified by Figure 1 and Figure 2 respectively.

Table 1: Antimicrobial activity of the Pergularia Tomentosa L. extracts against the test microorganisms.

	Diameter of Inhibition zone (mm)
	Ampicillin/ Nystatine	DMSO	Crude extract
Charge of the disc (µg) Bacteria strains	10μg/100μg	neat	20 mg/l
*Escherichia coli* ATCC 8739 G(-)	11.75±0.3	00	23
*Pseudomonas aeruginosa* G(-)	13.75±1.0	00	13
*Staphylococus aureus* ATCC 6538 G(+)	35.5±0.7	00	19

Fig 1: Microbial growth inhibition

Fig 2: The diameters of inhibition zone of Pergularia tomentosa L extract on the three types of tested gram negative and gram positive bacteria

The Minimum Concentration Inhibition (CMI) is defined as the lowest concentration of antibiotic to inhibit bacterial growth³³. It is grown in micro-pipettes from each bacterial suspension of 1μl at 104 UFC/ml. Petri dishes are placed in the incubator under 37°C for 24 hours, After 24 hours we read CMI where there is no clear growth of bacteria where we get the results listed in Table 2 and clarified by Figure 3 and Figure 4.

Table 2: CMI values of plant extract on the tested bacteria

CMI (mg/ml)	Bacteria strains
0.450	E. Coli
0.600	Staphylococcus aureus
1..25	Pseudomonas aeruginasa

Fig 3: CMI values of plant extract on the tested bacteria

Fig 4: Pictures of CMI values of plant extract on the tested bacteria

The results obtained in Table 3 indicate that the CMI values of the crude extract on E. coli, Staphylococcus aureus and Pseudomonas aeruginasa are respectively: 0.450; 0.600 and 1.25mg/ml.

Results for antibacterial activity as obtained with the crude extract of Pergularia tomentosa L. revealed that this extract exhibited a positive effect against two types of gram negative bacteria Escherichia coli ATCC 8739 G(-) and Pseudomonas aeruginosa G(-) and one gram positive Staphylococus aureus ATCC 6538 G(+), where the maximum activity was recorded against Escherichia coli ATCC 8739 G(-), with a maximum inhibition diameter of 23mm. Moreover the crude extract showed higher bacterial activity against Escherichia coli ATCC 8739 G(-) with an inhibition diameter of 23mm compared to the standard antibiotics Ampicillin/ Nystatine with an inhibition diameter of 11.75mm. On the other hand the DMSO solvent was ineffective with all bacteria strains tested Escherichia coli ATCC 8739 G(-),Pseudomonas aeruginosa G(-) and Staphylococus aureus ATCC 6538 G(+). These significant effects may be due to the extract effect on the permeability of the cell membrane and the function of the bacterial cell^34,35.

Generally, the extract of the plant is more or less effective towards the tested bacteria and is more potent compared to to the standard antibiotics Ampicillin/ Nystatine.

Antioxidant activity:

The anti-free radical activity was evaluated using DPPH, which was one of the first free radicals used to study the relationship structure antioxidant activity. 2,2-diphenyl-1-picrylhydrazil (DPPH), a stable, violet radical in solution and having a characteristic absorption maximum at 517nm. The protocol applied routinely relies on the disappearance of this maximum when the DPPH is reduced by a compound having antiradical property, thus causing discoloration towards the yellow color²³.

Antioxidant activity by using DPPH test:

This test is based on the ability of these extracts to inhibit 50% of the DPPH radicals and is calculated from the curves of the change in inhibition ratios in terms of concentration of extracts. The result is expressed in IC50.

We draw the graph curve of the inhibition in terms of concentrations. From the curves (Figure 5, 6 and 7), we obtained the appropriate concentration to eliminate 50% of the free radicals of the crude extract, Tocophorol (VE) and ascorbic acid (VC).

The effectiveness of crude flavonoid extract clarified by Figure 5 to trap the radical DPPH, expressed by the inhibition rate (I%) depending on the different concentrations. The values of the antioxidant capacity expressed in Tocopherol (VE) and ascorbic acid (VC) are determined and grouped together in Table 3.

Table 3: IC50 for tested extracts

Extract	Crude extract	Vitamin C	Vitamin E
IC50 (g/l)	0,012	0,010	0,015

Fig 5. DPPH Test of Tocopherol (VE)

Fig 6. DPPH Test of Ascorbic acid (VC)

Fig 7. DPPH Test of crude flavonoid extract

B-Antioxidant activity by using FRAP test:

Different concentrations of the extract in ethanol were prepared and each concentration of 100μl was treated in the same way as we treated tocoporol (VE):

100μl of each extract (+2ml of methanol +1 ml of FRAP mixture). At the same time, we conduct the same process on ascorbic acid (VC) used in the food industry in order to compare the results.

Using the standard curve Tocoforol (VE), we obtained the results shown in the Table 4.

Fig 8. FRAP Test of Tocopherol (VE)

Fig 9. FRAP Test of Ascorbic acid (VC)

Fig 10. FRAP Test of crude flavonoid extract

Among the three extracts, vitamin C is the most active with IC50 equal to 0.010g/ml, followed by the crude extract, with a value of 0.012g/ml and finally the Tocopherol (VE) which seems to be the least efficient with an IC50 of 0.015g/ml. It is clear through these values that antioxidant efficacy are used as preservatives in the food industry.

The equivalent antioxidant capacity for crude extract is 1.328g/l and 2.031g/l for ascorbic acid VC Table 4. These results show that ascorbic acid used in the industry as a preservative.

Table 4. The equivalent antioxidant capacity for crude extract and ascorbic acid VC

Extract	Crude extract	Vitamin C
VEAC (g/l)	1,328	2,031

In several reports, the antioxidant activity of the plants can be linked to the phenolic content. Indeed, the comparative study on the DPPH radical's reducing ability by different chemotypes has proved that the phenolic chemotypes had showed in-vitro, more expressed and much stronger antioxidant capacities than those of the non-phenolic chemotypes^36-37. In our case, the crude flavonoid extract is also rich with phenolic compounds and this plant has showed an antioxidant activity which sparks an interest for its therapeutic use and cosmetic applications.

CONCLUSION:

This study underscored the antimicrobial activity of Pergularia tomentosa L. This plant averred to be effective against two types of gram negative bacteria Escherichia coli G(-) and Pseudomonas aeruginosa G(-) and one gram positive Staphylococus aureus G(+), and the plant extract is more potent against Escherichia coli compared to the standard antibiotics Ampicillin/ Nystatine.

The study of the antioxidant activity by the DPPH and FRAP methods showed that vitamin C is the most active, followed by the crude extract, and finally the Tocopherol (VE) which seems to be the least efficient.

ACKNOWLEDGMENT:

The authors wish to express their sincere thanks to Algerian Ministry of Higher Education and Scientific Research (MHESR) for their support and providing the necessary facilities to carry out this research.

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Received on 19.03.2021 Modified on 20.04.2021

Asian Journal of Research in Chemistry. 2021; 14(4):285-291.

DOI: 10.52711/0974-4150.2021.00049